This invention relates to a chemical modification of biologically active interleukin-2 (IL-2) that alters the chemical and/or physiological properties of the IL-2 and an IL-2 mutein specifically designed for that purpose. More specifically, this invention relates to selective conjugation of IL-2 through its cysteine residues to polymers and to mutationally altered biologically active IL-2 muteins that differ from their parent analogs by the substitution of a particular amino acid with cysteine.
The use of polypeptides in circulatory systems for the purpose of producing a particular physiological response is well known in the medicinal arts. A limitation to the potential therapeutic benefit derived from the clinical use of polypeptides is their ability to elicit an immune response in the circulatory system. This immune response may be caused by aggregates in the material prior to injection as described by R. Illig (1970), J. Clin. Endrocr., 31, 679-688, W. Moore (1978), J. Clin. Endrocrinol. Metab., 46, 20-27 and W. Moore and P. Leppert (1980), J. Clin. Endrocrinol. Metab., 51, 691-697. This response involves the production of antibodies to the polypeptides by the circulatory system into which they are injected. This antibody production may decrease or eliminate the desired biological function of the polypeptide, sometimes by causing reduced residence time in the circulatory system (reduced half-life) or by modifying the molecule by virtue of the antibody-polypeptide interaction.
Modification of these potentially useful therapeutic polypeptides so as to preclude or at least reduce an immune response while still maintaining desired physiological activities of the polypeptide would allow the use of these polypeptides in the mammalian circulatory system without the aforementioned disadvantages. In addition, due to the increased half-life of the circulating polypeptide, smaller amounts of the polypeptide would be required for the desired therapeutic effect than have heretofore been possible.
The problems of immunogenicity and short half-life in circulation set forth hereinabove and other undesirable properties of certain proteins are well recognized and various methods have been undertaken to solve them. One includes the modification of proteins with substantially straight-chain polymers such as polyethylene glycol (PEG) or polypropylene glycol (PPG). For example, U.S. Pat. No. 4,261,973 describes conjugation of immunogenic allergen molecules with non-immunogenic water-soluble polymers such as PEG to reduce the immunogenicity of the allergen. U.S. Pat. No. 4,301,144 describes conjugation of hemoglobin to PEG, PPG, a copolymer of ethylene glycol with propylene glycol, or ethers, esters or dehydrated products of such polymers to increase the oxygen-carrying ability of the hemoglobin molecule. U.S. Pat. No. 4,609,546 discloses that conjugating of a polypeptide or glycoprotein to a polyoxyethylene-polyoxypropylene copolymer increases its physiological activity. Preferably the polypeptide or glycoprotein is an enzyme or native interferon, which are water soluble. U.S. Pat. No. 4,179,337 discloses conjugating of water-soluble polypeptides such as enzymes and insulin to PEG or PPG to reduce the immunogenicity of the polypeptide while retaining a substantial proportion of its desired physiological activity. U.S. Pat. No. 4,002,531 discloses a different method of conjugating enzymes to PEG through an aldehyde derivative.
U.S. Pat. No. 3,960,635 discloses pharmaceutical compositions comprising a water-soluble complex of a proteolytic enzyme linked covalently to a polymeric substance such as polysaccharides.
U.S. Pat. No. 3,960,830 discloses peptides bound to a polyalkylene glycol polymer such as polyethylene glycol.
U.S. Pat. No. 4,088,538 discloses a reversibly soluble enzymatically active polymer enzyme product comprising an enzyme covalently bonded to an organic polymer such as polyethylene glycol.
U.S. Pat. No. 4,415,665 discloses a method of conjugating an organic ligand containing at least one primary or secondary amino group, at least one thiol group and/or at least one aromatic hydroxy group (described in col. 3, lines 19-36) to a polymeric carrier with at least one hydroxyl group (described in col. 2, lines 42-66).
U.S. Pat. No. 4,496,689 discloses a covalently attached complex of alpha-1-proteinase inhibitor with a polymer such as PEG or methoxypolyethylene glycols.
U.S. Pat. No. 3,619,371 discloses a polymeric matrix having a biologically active substance chemically bound thereto.
U.S. Pat. No. 3,788,948 discloses use of organic cyanate compounds to bind proteins to polymers.
U.S. Pat. No. 3,876,501 discloses activation of water-soluble carbohydrates with cyanogen bromide to improve their binding to enzymes and other proteins.
U.S. Pat. No. 4,055,635 discloses pharmaceutical compositions of a proteolytic enzyme linked covalently to a polymeric substance.
EP 152,847 discloses an enzyme conjugate composition comprising an enzyme conjugate, a calcium salt, and a polyethylene glycol.
In addition to these patents and patent publications, several articles discuss the concept of using activated PEG or PPG as a modifying agent for proteins such as enzymes, IgG and albumin. For example, Inada et al., Biochem and Biophys. Res. Comm., 122, 845-850 (1984) disclose modifying water-soluble lipoprotein lipase to make it soluble in organic solvents such as benzene by using cyanuric chloride to conjugate with PEG. Takahashi et al., Biochem. and Biophys. Res. Comm., 121, 261-265 (1984) disclose modifying horseradish peroxidase using cyanuric chloride triazine with PEG to make the water-soluble enzyme active and soluble in benzene. Suzuki et al., Biochem. Biophys. Acta, 788, 248-255 (1984) disclose suppression of aggregation of IgG using cyanuric chloride activated PEG. Abuchowski et al., Cancer Biochem. Biophys., 7, 175-186 (1984) state that modification of asparaginases from E. coli and Vibrio succinogenes using PEG activated by succinimidyl succinate increases the half-life and decreases the immunogenicity of the proteins. Davis et al., Biomedical Polymers, (New York: Academic Press, 1980), p. 441-451 disclose that enzymes normally insoluble may be solubilized by PEG attachment without further details. Several other articles discuss modification of enzymes such as uricase, streptokinase, catalase, arginase and asparaginase with PEG activated by succinimidyl succinate or cyanuric chloride to increase half-life and decrease the immunogenicity of the protein.
None of these references, however, disclose specific IL-2 muteins obtained by site-directed mutagenesis and designed for conjugation to the PEG via an added cysteine residue nor conjugation of any IL-2 to PEG via cysteine residues. (U.S. Pat. No. 4,518,584 issued May 21, 1985 discloses IL-2 muteins wherein the cysteines are replaced by serines.) Furthermore, it is not a priori possible to predict which selected proteins would be favorably responsive to treatment with polymers due to the vast difference in the pharmacokinetics and physical properties of various proteins. Furthermore, none of the references disclose reducing or eliminating aggregation of the protein, a phenomenon that elicits an immune response when the protein is introduced in vivo.
EP 154,316, published Sep. 11, 1985 to Takeda Chemical Industries, Ltd., discloses and claims chemically modified lymphokines such as IL-2 containing PEG bonded directly to at least one primary amino group of a lymphokine.
In addition, WO 86/04145, published Jul. 17, 1986 to University of New Mexico discloses covalent modification of proteins with PEG employing an active ester intermediate.
Finally, PCT W087/00056 published Jan. 15, 1987 (Cetus Corporation) discloses that IL-2, IFN-.beta. and immunotoxins may be conjugated to PEG or polyoxyethylated polyols by means of lysines or cysteines on the protein.